The Role And Mechanism Of LncRNA TCONS-00106987 In Atrial Electrical Remodeling Of Experimental Atrial Fibrillation

BackgroundsAtrial fibrillation(AF)is the most common tachyarrhythmia in clinic,with high morbidity and mortality.The epidemiological study of Framingham showed that the prevalence rate of AF in foreign population reaches 0.5%,the prevalence rate of people over 60 years old is 6%,and the prevalence rate of people over 80 years old is as high as 8.8%.A cross-sectional survey in 2018 found that the prevalence of AF reached 0.71%among china residents over 35 years old.Long-term AF can cause serious complications.Studies have shown that the incidence of heart failure in patients with AF is 3 times higher than that in normal people,and the risk of stroke is 5 times higher.At present,the main clinical treatments for AF include drug therapy,maze surgery,radiofrequency ablation and so on,but these therapies have varying degrees of side effects and a relatively high recurrence rateAF is a disease caused by many factors,and its pathogenesis is complicated.At present,it is believed that atrial remodeling is an important basis for the occurrence and maintenance of AF.Atrial remodeling includes electrical remodeling,structural remodeling and nerve remodeling.In the early stage,AF is mainly manifested by the changes of ion channels and electrophysiology,that is,atrial electrical remodeling.The changes of ion channels mainly include the decrease of L-type calcium current(ICAL),the increase of inward rectifier potassium current(IKI)and the abnormal distribu)f half-channel gap junction.The main electrophysiological changes were shortening of atrial effective refractory period(ERP)and action potential duration(APD).In-depth study of the mechanism of atrial electrical remodeling in AF is helpful to further improve the prevention and treatment of AF.Long non-coding RNA(lncRNAs)is a class of heterogeneous transcripts length from 200nt to 100kb with no protein coding potential.A large number of studies have shown that lncRNAs may be involved in a variety of cell processes,including proliferation,differentiation,cell cycle regulation and apoptosis.From a functional point of view,lncRNAs participates in gene expression and cellular activities through a variety of mechanisms,including chromatin remodeling,binding to transcription factors,competitive binding to microRNAs(miRNAs),alternative splicing of mRNA and translational regulation.Studies have found that lncRNAs regulatory disorders are associated with many heart diseases,including myocardial infarction,cardiac dysfunction and heart failure,suggesting that they may become therapeutic targets and/or biomarkers.However,there are relatively few studies on the potential role and mechanism of lncRNAs in AF.The purpose of this study is to explore the roles and mechanisms of lncRNAs in AF.The study is divided into two parts.In the first part,the rabbit model of AF was established by rapid pacing,and the second generation high-throughput sequencing was used to screen out the differentially expressed lncRNAs,bioinformatics analysis was applied to screen out lncRNA related to atrial electrical remodeling in AF.Then we studied the role of target lncRNA in AF through in vitro overexpression and silencing experiments.The second part deeply studies the mechanism of the role of IncRNA in atrial electrical remodeling of AF,and explains the mechanism of the IncRNA on atrial remodeling through in vivo and in vitro experiments.The purpose of this study is to provide reference for the molecular pathogenesis of AF and to find potential therapeutic targets,and to promote the research of lncRNAs related to the pathogenesis of AF.Part one:Roles of IncRNAs in experimental atrial fibrillation models.Methods1.Twelve healthy adult New Zealand rabbits were randomly divided into two groups:AF group and control group.In the AF group,a pacemaker was implanted into right atria via jugular vein for continuous rapid pacing,while in the control group,only pacemakers were implanted without pacing.7 days later,the atrial effective refractory period(ERP)and the induction rate of AF were measured2.The differentially expressed lncRNAs was screened by the second generation high-throughput sequencing.The total RNAs of rabbit atria was extracted for the second generation high-throughput sequencing.The expression of mRNAs and lncRNAs in each group was detected,and the significant differentially expressed mRNAs and lncRNAs were screened out.3.The differentially expressed lncRNAs and mRNAs were analyzed by GO(Gene Ontology)enrichment analysis and KEGG(Kyoto Encyclopedia of Genes and Genomes)pathway analysis,and on this basis,target gene prediction,coexpression analysis,tissue specificity analysis and other bioinformatics analysis were performed to screen the target lncRNA related to atrial electrical remodeling4.The shRNA targeting lncRNA TCONS-00106987 was cloned into lentivirus expression vector and lentivirus overexpress lncRNA TCONS-00106987 was also synthesized.Negative control lentivirus was used to control off-target effect and non-specific effect.Fifteen New Zealand white rabbits were randomly divided into 3 groups After thoracotomy,1 × 107 corresponding virus particles were injected into 10 different parts of right atria.AERP and the induction rate of AF were measured before infection and 14 days after infection.The changes of atrial electrophysiology before and after lentivirus infection were detectedResults1.The rabbit models of AF were successfully established by rapid atrial pacing.Compared with the control group,seven days after pacing,the atrial ERP of AF group was significantly shortened,the induction rate of AF was significantly increased2.Through the second generation of high-throughput sequencing,1364 differentially expressed lncRNAs were screened out,of which 1110 transcripts were down-regulated and 254 transcripts were up-regulated.And 956 genes were differentially expressed,of which 395 genes were down-regulated and 561 genes were up-regulated.We applied GO and KEGG analyses to differentially expressed genes,and predicted the target gene of lcnRNAs found that the expression of IncRNA TCONS-00106987 was significantly upregulated in AF group,and it was speculated that lncRNA TCONS-00106987 was related to atrial electrical remodeling.LncRNA TCONS-00106987 was selected as the target IncRNA for in-depth study.3.After infection of lentivirus overexpression IncRNA TCONS-00106987,lentivirus silencing lncRNA TCONS-00106987 and negative control lentivirus into rabbit atria,compared with the negative control group,atrial ERP in the lncRNA TCONS-00106987 overexpression group was significantly shortened and the induction rate of AF was significantly increased,while atrial ERP in the lncRNA TCONS-00106987 inhibition group was extended,and the induction rate of AF was loweredConclusions1.The electrophysiology of right atria in AF rabbits is altered,and IncRNAs have a specific differential expression profile in AF;2.Differentially expressed mRNAs in AF enriched in specific functions and pathways.The differentially expressed IncRNAs exist co-expression with mRNAs,indicated that lncRNAs may participate in AF through various patterns3.The expression of lncRNA TCONS-00106987 is increased in the atria of rabbits with experimental AF.Overexpression of lncRNA TCONS-00106987 can shorten atrial ERP,and increase the induction rate of AF.It indicated that lncRNA TCONS-00106987 can promote atrial electrical remodeling in the process of AF.Part two:The mechanism of lncRNA TCONS-00106987 modulates atrial electrical remodeling in atrial fibrillation.Methods1.Bioinformatics analysis of lncRNA TCONS-00106987To find the target genes and function mechanisms of lncRNA TCONS-00106987,we applied cis-,trans-prediction and database analysis to the sequence of IncRNA TCONS-001069872.Dual luciferase reporter systemThe wild type and mutant type of the possible binding sites between lncRNA TCONS-00106987/miR-26 and KCNJ2/miR-26 were cloned into luciferase vector.The luciferase vector was cotransfected with miR-NC or miR-26 mimics respectively,and the signal value of luciferase in each group was detected3.Overexpression and silencing of IncRNA TCONS-00106987 in vitroThe primary cardiomyocytes were randomly divided into three groups:negative control group,lncRNA TCONS-00106987 overexpression group and lncRNA TCONS-00106987 silencing group.After infection of corresponding lentivirus,RNA and protein were extracted to detect the expression of target genes4.Co-infection of lncRNA TCONS-00106987 and miR-26 overexpression lentivirus in vivo and in vitroLncRNA TCONS-00106987 overexpression lentivirus and miRNA-26 overexpression lentivirus were co-infected into primary cardiomyocytes and rabbit right atria.The changes of atrial electrophysiology,target gene and current density were detected before and fourteen days after infectionResults1.Through the database comparison of the full length sequence of IncRNA TCONS-00106987,we found that KCNJ2 was the neighbor gene of IncRNA TCONS-00106987 The expression of KCNJ2 was regulated after overexpressing and depressing of lncRNA TCONS-00106987,which indicated that KCNJ2 is the target gene of IncRNA TCONS-00106987.2.LncRNA TCONS-00106987 and KCNJ2 shared the same binding site with miR-26.Dual luciferase reporter system results showed that miR-26 inhibited the signals produced by the wild type reporter genes of IncRNA TCONS-00106987 and KCNJ23’UTR,while the mutant type reporter genes signals of TCONS-00106987 and KCNJ23’UTR were not affected.The results indicated that miR-26 can function through binding with the predicted sites.3.Compared with the negative control group,there was no significant increasing in the expression of KCNJ2 mRNA and protein,no significant shortening of atrial ERP and no significant increasing in the induction rate of AF in the co-infection group.In primary cardiomyocytes co-infected with lncRNA TCONS-00106987 overexpression and miR-26 overexpression lentivirus,the expression of KCNJ2 mRNA and protein were not significantly up-regulated,and IKl density was not significantly increased.These results revealed that lncRNA TCONS-00106987 can competitively binding with miR-26 to regulate the expression KCNJ2 and modulate atrial electrical remodeling.Conclusions1.Electrical remodeling related gene KCNJ2 is the target gene of lncRNA TCONS-00106987,the expression of KCNJ2 is affected by lncRNA-TCONS00106987.The analysis shows that lncRNA TCONS-00106987 may reduce the inhibitory effect of miR-26 on KCNJ2 by competitively binding with miR-26,thus up-regulate the expression of KCNJ2 and promote atrial electrical remodeling.2.The results of dual luciferase reporter system showed that the binding sites between lncRNA TCONS-00106987/miR-26 and miR-26/KCNJ2 were the direct functional target of lncRNA TCONS-00106987.3.Overexpression of miR-26 can reverse the upregulation of KCNJ2 expression and atrial electrophysiological changes caused by overexpression of lncRNA TCONS-00106987.It is confirmed that lncRNA TCONS-00106987 can promote atrial electrical remodeling through competitively binding with miR-26 to decrease the inhibition effect of miR-26 on KCNJ2 in vivo and in vitro,resulting in AF.